Chip based DNA microarrays are an integration of circuit fabrication technology and genetics. DNA microarrays consist of matrices of DNA arranged on a solid surface where the DNA at each position recognizes the expression of a different target sequence. Microarrays are used to identify which genes are turned on or off in a cell or tissue, and to evaluate the activity level under various conditions. This knowledge enables researchers to determine whether a cell is diseased or the effect of a drug on a cell or group of cells. These studies are critical to determine a drug's efficacy or toxicity, to identify new drug targets, and to more accurately diagnose illnesses, such as specific types of cancer. The technology is useful to classify tumors with the hope of establishing a correlation between a specific type of cancer, the therapeutic regiment used for treatment, and survival.
Expression microarrays are used to detect the presence of nucleic acids or polynucleotides generated, or expressed, by genes. These nucleic acids, or “targets,” may be taken from any biological source, including healthy or diseased tissue, tissues that have been exposed to drugs, and pathogens. Because expression microarrays are often used to determine if a tissue is expressing different biomolecules than normal due to disease or drug treatment, the targets of interest are often nucleotides produced by these tissues.
Generally, single nucleotide polymorphism (SNP) microarrays are similar to expression microarrays, including their use of oligonucleotide probes and nucleic acid targets. However, differences can exist regarding how fluorescent labels are attached to the targets and how the microarrays are developed.
Another type of microarray, proteomic or protein arrays, are used to measure protein levels in cells. Generally, these arrays use antibodies as probes and cell lysates as targets. They are useful in high-throughput protein discovery, protein profiling, protein structure, and activity analyses, as well as protein-protein and protein-small molecule interaction studies.
Microarrays typically have biomolecules (probes) such as oligonucleotides or polypeptides attached to a polymer-coated solid support and arranged such that each of many small regions on the surface of the microarray contains a biomolecule that is at least slightly different from that of another region. When the microarray is contacted with a sample under appropriate conditions, components of the sample (targets) may bind to one or more biomolecules on the microarray. In order to detect the bound targets, the target typically contains a fluorescent molecule or dye that fluoresces when irradiated with light at its excitation wavelength. Fluorescent molecules are commonly referred to a “fluorophores” or “labels.”
Multiple methods exist to incorporate fluorescent molecules into targets (See U.S. Pat. No. 6,203,989). A commonly used process is referred to as Tyramide Signal Amplification (TSA), which may be used to detect low concentrations of a molecule within a sample (See U.S. Pat. Nos. 5,731,158; 5,583,001; and 5,196,306).
Typically, during a TSA amplification, biotin is incorporated into the target of interest. Alkaline phosphatase (AP) or horseradish peroxidase (HRP) enzyme bound streptavidin or avidin is added. Tyromide labeled with a fluorophore is then added. The AP or HRP enzyme then incorporates multiple labeled tyramides at the probe site, thereby increasing or amplifying the number of fluorophores at the probe/target site.
TSA methods generate a large number of fluorescent labels at the probe/target site due to the enzymatic action of the AP or HRP. Thus, the actual number of fluorophores at the probe/target site is not linearly related to the number of probe/target bindings which occur, but is dependent on the activity of the enzymes.
When microarrays are formed using hydrogels as the solid support coating, TSA methods result in large levels of background noise, thus providing a poor signal to noise ratio. Such high levels of background noise are likely a product of variation in the enzymatic reaction. Therefore, there is a need for high-sensitivity target detection methods for use with hydrogel microarrays that provide a good signal to noise ratio.